Fig. 5: PYCR1-catalyzed NAD⁺ production is involved in transcriptional regulation.

a, d mRNA levels of indicated ELK4 target genes were analyzed by real-time PCR. In b and e-g, the primers covering the ELK4-binding sites at promoter regions of the indicated genes were utilized for real-time PCR analysis. The y axis shows the value normalized to the input. a, b, d–g data are presented as the mean ± s.d. (n = 4 independent experiments); statistical analysis was performed using the two-tailed Student’s t test. Source data are provided as Source Data Fig. 5. a, b HCT116 cells expressing the indicated Flag-rPYCR1s were simultaneously overexpressed with or without NMNAT-1. Cells were supplemented with the metabolites as indicated. Cells were cultured under hypoxia for 24 h (a) or 12 h (b). Real-time PCR (a) or ChIP (b) analysis was performed. c The cartoon showing the key enzymes involved in proline synthesis from glutamine. d, e HCT116 cells with or without P5CS depletion were cultured under normoxia or hypoxia for 24 h (d) or 12 h (e). Real-time PCR (d) or ChIP (e) analysis was performed. f, g HCT116 cells with depleted P5CS (f) or with expression of rPYCR1 T238A (g) were transfected with or without Sirt7 siRNA. Cells were cultured under hypoxia for 12 h. Chromatin extracts were collected and mixed with or without the indicated metabolites for 30 min, and then chromatin extracts were fixed and used for ChIP analysis.